To begin sample preparation for flow cytometry, take out stimulation tubes containing whole blood samples with Brefeldin A from the incubator. Add 500 microliters of 0.5 molar EDTA solution to each stimulation tube containing Brefeldin A.Transfer the samples into new 15 milliliter centrifuge tubes. Then pipette one milliliter of erythrocyte lysis buffer into each stimulation tube to rinse it.
Transfer the buffer and cell suspension into the same centrifuge tube. Centrifuge the tubes at 600 g for seven minutes, and carefully pipette out the supernatant. Then add erythrocyte lysis buffer to the pellet and resuspend it.
Incubate samples at room temperature until the samples appear clear or for a maximum of six minutes. Centrifuge the tubes at 600 g for seven minutes. After discarding the supernatant, add one milliliter of HBSS to the pellet.
Transfer the cell suspension into two-milliliter reaction tubes. Then centrifuge the tubes at 400 g for five minutes. Discard the supernatant before performing flow cytometric staining.
Transfer the samples from the stimulation tubes that do not contain Brefeldin A into 1.5 milliliter tubes. Centrifuge the tubes at 2, 000 g for 20 minutes. Now carefully pipette the supernatant into a fresh 1.5-milliliter tube.
If not analyzing the supernatant immediately, cryopreserve the sample at 80 degrees Celsius. When using frozen samples, centrifuge thawed supernatants again at a speed greater than 7, 000 g for five minutes before analysis. Perform pre-dilution of the samples as required by the cytokine assay protocol.
Resuspend the cell pellet in one milliliter of RNA protection buffer. Cryopreserve it at 80 degrees Celsius for subsequent RNA isolation. Alternatively, resuspend the cell pellet in 700 microliters of lysis buffer for immediate RNA isolation.
